Biological monitoring is a core element in the water resource management and in the conservation of ecological integrity in water ecosystems (Karr, J. R. 1991. Biological integrity: a long-neglected aspect of water resource management. Ecological Applications, 1: 66-84; Rosenberg, D. M., and Resh V. H. (Eds.). 1993. Freshwater biomonitoring and benthic macroinvertebrates. Chapman and Hall (Eds.), New York, 488p; Karr, J. R., and Chu, E. W. 1999. Restoring Life in Running Waters: Better Biological Monitoring. Island Press, Washington, D.C.).
Biological water ecosystem monitoring programs were created in the early XX century by KOLKWITZ & MARSSON ([Kolkwitz, R., and Marsson, M. 1908. Ökologie der pflanzlichen Saprobien. Bericht der Deutschen Botanischen Gesellschaft 26a: 505-519. (Translated 1967). Ecology of plant saprobia. In Kemp, L. E., W. M. Ingram & K. M. Mackenthum (eds), Biology of Water Pollution. Federal Water Pollution Control Administration, Washington, D.C.: 47-52.] [Kolkwitz, R., and Marsson, M. 1909. Ökologie der tierischen Saprobien. Beiträge zur Lehre von des biologischen Gewasserbeurteilung. Internationale Revue der gesamten Hydrobiologie und Hydrographie, 2: 126-152.]), which set the conceptual foundations for the construction of biomonitoring methods.
From their inception to the end of the 1980s, biotic indices predominated as biological monitoring tools ([Metcalfe, J. L. 1989. Biological Water Quality Assessment of Running Waters Based on Macroinvertebrate Communities: History and Present Status in Europe. Environmental Pollution, 60: 101-139.]; [Rosenberg, D. M., and Resh V. H. (Eds.). 1993. Freshwater biomonitoring and benthic macroinvertebrates. Chapman and Hall (Eds.), New York, 488p)].
More recently, new approaches were set as tools for biomonitoring such as predictive models (RIVPACS—UK; AusRivAs—Australia; BEAST—Canada, New Zealand model) (Wright, J. F. 1995. Development and use of a system for predicting the macroinvertebrate fauna in flowing waters. Australian Journal of Ecology 20: 181-197; Norris, R. H., and Georges, A. 1993. Analysis and interpretation of benthic macroinvertebrate surveys. Chapman and Hall, New York (USA), pp. 234-286. 1993; Reynoldson, T. B.; Bailey, R. C; Day, K. E., and Norris, R. H. 1995. Biological guidelines for freshwater sediment based on Benthic Assessment of SedimenT (the BEAST) using a multivariate approach for predicting biological state. Australian Journal of Ecology 20:198-219; Joy, M. K., and Death, R. G. 2003. Biological assessment of rivers in the Manawatu-Wanganui region of New Zealand using a predicative macroinvertebrate model. New Zealand Journal of Marine and Freshwater Research 37: 367-379).
The development of multimetric indices has been prioritized in the US since the late 1980s ([Plafkin, J. L.; Barbour, M. T.; Porter, K. D.; Gross, S. K., and Hudges R. M. 1989. Rapid bioassessment protocols for use in sites and rivers: Benthic macroivertebrates and fish. U.S. Environmental Protection Agency, EPA, 444/4-89-001, Washington, D.C.], [Barbour, M. T.; Gerritsen, J.; Griffith, G. E.; Frydenborg, R.; McCarron, E.; White, J. S., and Bastian, M. l. 1996. A framework for biological criteria for Florida streams using macroinvertebrates. Journal of North American Benthology Society. 15 (2), 185-211]; [Barbour, M. T.; Stribling, J. B., and Karr, J. R. 1995. The multimetric approach for establishing biocriteria and measuring biological condition. Pp: 63-76. In: Davis, W. & Simon, T. (eds). Biological Assessment and Criteria: Tools for Water Resource Planning and Decision Making.] [Lewis Publishers. Ann Arbor, Mich.; Barbour, M. T.; Gerritsen, J.; Griffith, G. E; Frydenborg, R.; McCarron, E.; White, J. S., and Bastian, M. L. 1996. A framework for biological criteria for Florida sites using benthic macroinvertebrates. J. N. Am. Benthol. Soc, 15(2): 185-211)]; [Gibson, G. R.; Barbour, M. T.; Stribling, J. B.; Gerritsen, J., and Karr, J. R. 1996. Biological Criteria. Technical Guidance for Sites and Small Rivers. EPA/822-B-96-001. U.S. Environmental Protection Agency. Office of Science and Technology, Washington, D.C.]). European Union countries recently started to invest in the standardization and use of multimetric indices, following the proposals set by the Water Framework Directive No. 2000/60/EC (EC, 2000 European Commission. The EU Water Framework Directive—Integrated River Basin Management for Europe. Available at: http://ec.europa.eu/environment/water/water-framework/index_en.html accessed on: Feb. 21, 2008.). In this sense the EU produced the AQEM and STAR projects to standardize and inter-calibrate the operating procedures and development of different multimetric indices, based on the fauna of macroinvertebrates (Pinto P.; Rosado, J.; Morais, M., and Antunes, I. 2004). Assessment methodology for southern siliceous basins in Portugal. Hydrobiology, 516: 193-216; Bohmer, J.; Rawer-Jost, C, and Zenker, A. 2004. Multimetric assessment of data provided by water managers from Germany: assessment of several different types of stressors with macrozoobenthos communities. Hydrobiologia, 516: 215-228; Vlek, H. E.; Verdonschot, P. F. M., and Nijboer, R. C. 2004. Toward a multimetric index for assessment of Dutch stream using benthic macroinvertebrates. Hydrobiologia, 516: 173-189; Buffagni, A.; Erba, S.; Cazzola, M., and Kemp, L. L. 2004. The AQEM multimetric system for the southern Italian Alpennines: assessing the impact of water quality and habitat degradation on pool macroinvertebrates in Mediterranean rivers. Hydrobiologia, 516: 313-329; Furse, M. T.; Hering, D.; Brabec, K; Buffagni A.; Sandin, L., and Verdonschot, P. F. M. 2006. The Ecological Status of European Rivers: Evaluation and Intercalibration of Assessment Methods. Hydrobiologia, 566: 3-29).
The strength of the multimetric approach lies in the ability to integrate data from the various aspects of a community to provide a general classification of the level of degradation in an ecosystem without losing information from individual metrics. The metrics should be based on solid ecological concepts and represent complex ecosystem processes, to allow for the assessment of ecological functions. The use of different nature metrics may allow for a qualitative evaluation, in addition to the quantitative one, as a metrics may, individually, be able to qualify the source of the impact.
In general, all of the indices were initially formulated considering exhaustive collection and separation work in the surveying of the macroinvertebrate benthic fauna. Therefore, the indices are constructed considering a biological database that is very robust but with limited application in routine procedures.
From a practical standpoint, following the collection procedure, all the substrates sampled, organic materials (leaves/algae/macrophytes) and minerals (silt, sand, fine rock, stones) are transported to the laboratory and washed and after that the separation and identification of the biological material are initiated; it should be highlighted that the volume of raw material collected can reach up to 15-20 liters. Among the disadvantages of these techniques we could point the large volumes of the samples collected that have to be correctly treated and stored, the time spent in separating the substrate and the sizable amount of hours spent in the identification of all the specimens, apart from the large quantity of alcohol used in the preservation of the material. We should also point that the number of specimens collected frequently reaches thousands of larvae, which considerably increases operating costs and the environmental impact.
In this context quick evaluation protocols are being developed as simple tools and with low application costs, to assess the health of water ecosystems. These protocols blend simple and cost-effective field equipment with an optimized processing of the samples in the lab.
Subsampling is a technique currently used in Europe and in the US, consisting of counting and identifying a part of the randomly obtained community in the total sample collected in the field. The goal of subsampling is to generate a faithful and unbiased representation of a larger sample. It should be random and incorporate the heterogeneous character and diversity of the habitats studied in the field. This leads to a reduction of the effort required.
With this system, all the material collected is taken to the lab, washed and mixed through different techniques, allowing it to become homogeneous. Through a subsampler (tray split into 24 areas) one randomly chooses a portion of the sample
Quick evaluation protocols produced in the US ([Plafkin, J. L.; Barbour, M. T.; Porter, K. D.; Gross, S. K., and Hudges R. M. 1989. Rapid bioassessment protocols for use in sites and rivers: Benthic macroinvertebrates and fish. U.S. Environmental Protection Agency, EPA, 444/4-89-001, Washington, D.C.], [Barbour, M. T.; Gerritsen, J.; Snyder, B. D.; and Stribling, J. B. 1999. Rapid Bioassessment Protocols for Use in Sites and Rivers: Periphyton, Benthic Macroinvertebrates and Fish, Second Edition. EPA 841-B-99-002. The US Environmental Protection Agency; Office of Water; Washington, D.C.]) traditionally recommend subsampling via counting of a fixed number. In these protocols the minimum number of organisms recommended to ensure efficiency in evaluation is of at least 300 individuals; in order to prevent much instability in the index metrics and provide reliable results for the evaluation. In practical terms, however, there is a big variation in the minimum number of organisms counted, depending on the analysis at hand. Additionally, when comparing the number of subsamples, it is possible to see the frailty in the small amount of samples.
Another type of subsampling is that done per area which is also the standard procedure suggested by the AQEM. This protocol suggests the use of trays split into quadrats where 25% of the total sample, of a minimum 300 individuals, are sorted. Area subsampling guarantees the random nature of the procedure, making it less subjective and less prone to the variations inherent to team change. However, there are still problems related to the large volume of the samples collected, to their storage, conservation, separation from the substrate, amount of alcohol used, and the quantity of specimens collected, that can reach thousands of individuals, amongst larvae and adults.
Regardless of the kind of sampling, the existing state-of-the-art methods have been discussed in several studies in countries where biomonitoring programs are already in application (EU, Australia and the US) ([Barbour, M. T.; Gerritsen, J.; Griffith, G. E.; Frydenborg, R.; McCarron, E.; White, J. S., and Bastian, M. l. 1996. A framework for biological criteria for Florida streams using macroinvertebrates. Journal of North American Benthology Society. 15 (2), 185-211]; [Countermanch, D. L. 1996. Commentary on the subsampling procedures used for rapid bioassessments. Journal of North American Benthological Society 15: 381-385]; [Somers, K. M.; Reid, R. A., and S. M. 1998. Rapid ecological assessment: how many animals are enough. Journal of the North American Benthological Society 17: 348-358.]; [Doberstein, C. P.; Karr, J. R.; Conguest, L. L. 2000. The effect of fixed-count subsampling on macroinvertebrate biomonitoring in small streams. Freshwater Biology, Volume 44 (2): 355-371]; [Lorenz, A.; Hering, D.; Feld, C, and Rolauffs, P. 2004. A new method for assessing the impact of hydromorphological degradation on the macroinvertebrate fauna of five German stream types. Hydrobiologia, 516: 107-127]).
One of the biggest issues associated with biosampling is that of the wealth of species. The number of taxa found in a sample increases asymptotically as a function of the area sampled and of the number of individuals in the sample. Thus, it is always expected that, with the increase in the effort, one would obtain a greater wealth of species. The issue to focus on, in the specific case of subsampling for biomonitoring is that when this increase no longer is significant and, at the same time, provides an explanation for the change in ecosystem integrity. Apart from that, the full processing of this type of sample, with many individuals, is too costly.
Thus, the state-of-the-art is embedded with the issue is of how to carry out the subsampling and what the optimal effort is, in the sense of speeding the evaluation without impairing the ecological validity of the response ([Barbour, M. T.; Gerritsen, J.; Griffith, G. E; Frydenborg, R.; McCarron, E.; White, J. S., and Bastian, M. L. 1996. A framework for biological criteria for Florida sites using benthic macroinvertebrates. J. N. Am. Benthol. Soc, 15(2): 185-211]; [Countermanch, D. L. 1996. Commentary on the subsampling procedures used for rapid bioassessments. Journal of North American Benthological Society 15: 381-385]; [Doberstein, C P.; Karr, J. R.; Conguest, L. L. 2000. The effect of fixed-count subsampling on macroinvertebrate biomonitoring in small streams. Freshwater Biology, Volume 44 (2): 355-371]; [Nichols, S. e Norris, R. H. 2006. River condition assessment may depend on the sub-sampling method: field live-sort versus laboratory sub-sampling of invertebrates for bioassessment. Hydrobiologia, 572: 195-213]). The subsampling should preferably be carried out in the field or, better yet, in the laboratory.
Clarke and collaborators (2006) (Clarke, R T.; Furse, M T.; Gunn, R. J. M.; Winder, J. M., and Wright, J. F. 2002. Sampling variation in macroinvertebrate data and implication for river quality indices, Freshwater Biology 47: 1735-1751) studied the effect of subsampling directly on the metrics of different types and found that the precision of the measurements based on the wealth of taxa is quite affected by the size of the subsample, which is predictable due to the species-area ratio.
Apart from the analysis of the sampling effort, it is always necessary to verify if the subsampling apparatus guarantees the randomization of the organisms, that is, that the organisms are in a given quadrat by chance. A trend observed in this stage can lead to errors in determining the minimum evaluation effort and, in the context of a biomonitoring program, errors in the evaluation of ecological integrity. In biological terms, it is necessary to ask whether the organisms are randomly distributed in the space, or in this case, in the subsampling tray. If the random pattern indeed exists, the Poisson distribution is the right statistical descriptor for the data (Krebs, C. J. 1998. Ecological Methodology. Benjamin/Cummings, Menlo Park.). The Poisson distribution assumes that the expected number of organisms of a particular taxon is the same in all the quadrats and is equal to the population average, estimated based on the sampling average.
In this context, several subsamplers are found in the state-of-the-art. They basically consist of a plastic tray split into 24 areas. This equipment allows for the reduction of relative time in substrate separation and fauna identification, but does not solve the issues related to the large volume of samples collected, their storage, conservation, amount of alcohol used and the number of specimens collected. However, on the other hand, they produce damage to the specimens as a result of the homogenizing process that hamper the separation and identification, apart from not contributing to the preservation of the biota.
It is important to point that, if time and resource-saving procedures such as subsampling are applied to the biological monitoring with no prior analysis for equipment accuracy and precision, as well as methods used, the data collected could be useless, resulting in waste of resources, or even in the misled application of handling measures. On the other hand, the application of exhaustive procedures that use much lab time and resources, taking long to provide the biological answer are not practical in terms of application of biomonitoring programs that should assess the condition of hundreds of water bodies. Thus, equipment and methodologies are needed that would allow for an ideal cost-benefit ratio, ensuring the applicability of the tool, without the loss of scientific rigor and power to inference and decision.
This way, the creation of new subsampling equipment and methodologies that gather the usability features for small volumes, random distribution of the fauna, maintenance of their integrity, and environmental respect, are needed.